In:
"Probing Photosynthesis: Mechanisms, Regulation And
Adaptation", edited by Mohammad Yunus, Uday Pathre, and
Prasanna Mohanty, published in 2000by Taylor & Francis,
London, pp.9-39. A
pdf file of the chapter and a correct Figure 3 is available by
sending an E-mail to gov@uiuc.edu

GOVINDJEE
Departments of Biochemistry, Plant Biology and Center of
Biophysics and Computational Biology
University of Illinois at Urbana-Champaign

The task of writing a chapter on milestones in photosynthesis
research is difficult. because there are so many milestones that
I may not be able to do justice to them all. Thus, at the very
outset, I beg forgiveness for incompleteness and I urge the
readers that they should not consider this chapter as a record of
the history of photosynthesis. Further, this chapter will not
present milestones (including the ones marking one tenths of a
mile) in a linear chronological manner, but according to
specified topics. For an earlier thought-provoking article on the
conceptual development in photosynthesis, see Myers (1974), for
historical development, see Huzisige and Ke (1993), and for an
introductory overview on photosynthesis, see Whitmarsh and
Govindjee (1995).An overview on the milestones in the
area of chlorophyll a fluorescence has been presented
earlier (see Govindjee, 1995; also see Govindjee et al., 1986,
and the chapter by Strasser and coauthors, this volume); thus, it
will not be covered here unless I consider it pertinent to my
discussion.

A tribute.This chapter is written in honor of my dear friend, a trusted
colleague and a World leader in the field of Photosynthesis, Dr.
Prafullachandra Vishnu Sane. I refer here only to three of his
contributions. A thought-provoking new model of the distribution
of the two photosystems in the thylakoid membrane system was
presented elegantly by him in collaboration with Rod Park in
their classic review (Park & Sane, 1971; also see Sane,
1977). His pioneering research, in collaboration with V.G. Tatake
& T.S. Desai, in devising methods to record the most highly
resolved thermoluminescence bands from the photosynthetic
material, and for assigning these bands not only to photosystem
II, but also photosystem I, have been reviewed by him in a
chapter in a book (see Sane & Rutherford, 1986). I would also
like to mention his discovery of heat-induced "state
changes" in photosynthetic systems (see Sane et al., 1984).

The primary source of energy for nearly all life on Earth is
the Sun. As early as 1845, Robert Mayer, who provided us with the
Law of Conservation of Energy, had already recognized that plants
convert light energy into chemical energy on a massive scale.
Photosynthesis is the physico-chemical process by which oxygenic
(plants, algae, cyanobacteria and prochlorophytes) and anoxygenic
(photosynthetic bacteria) organisms convert light energy into
redox chemical energy on a global scale. Each year 4x1018
kilojoules of free energy is stored in reduced Carbon by this
process. In terms of carbon, each year about 1011 metric tons of
CO2 are converted into organic matter by photosynthesis.
According to Woese et al. (1990) the living organisms can be
divided into three groups: archea; bacteria; and eukarya.
Archea do not engage in true photosynthesis although a
bacteriorhodopsin-containing organism Halobacterium salinarium
(formerly H.halobium) can convert light energy
into adenosine tri-phosphate (ATP). Photosynthetic bacteria and
cyanobacteria, mentioned above, are clearly bacteria as
the name implies, whereas plants and algae are eukarya.
Oxygenic photosynthesis provides us with both food and oxygen,
and anoxygenic photosynthesis only with food, needed for the
survival of almost all living organisms except certain bacteria
(two examples are Methanococccus janaschii and Methanobacterium
autotrophicum). In addition, ancient photosynthesis is still
providing us with fossil fuel; at the rate we are using it, it is
not going to last forever. We need to understand the basics and
the historical development of photosynthesis since it is the only
process that can provide us with food, fuel and oxygen needed for
the ever-increasing population, and since it is the only major
process that may utilize the global increases in CO2 we are
experiencing. It is now obvious that by the year 3,000, we expect
the World's population to be 13 billion, but at the same time the
available land for food production/person is decreasing at an
alarming rate caused by our follies (increased deforestation and
fossil-fuel burning) (Kendall et al., 1997; Sinha &
Swaminathan, 1991; Swaminathan, 1998). Drastic measures are
required to overcome this impending crisis that is already on our
doorstep!

Within eight minutes of its origin, sunlight reaches the
photosynthetic organisms on the Earth; almost billion chlorophyll
molecules in a single chloroplast function to capture this energy
within femtoseconds (10-15 s) (Note just for fun: there are as
many femtoseconds in a second as there are seconds in 31 million
years.) In what follows, I present a historical and a conceptual
perspective on the milestones and breakthroughs in photosynthesis
research dealing with the conversion of light energy into
chemical energy in the form of reduced nicotinamide adenine
dinucleotide phosphate (NADPH) and adenosine tri phosphate (ATP),
which leads to the production of food from CO2 (see a general
review by Whitmarsh & Govindjee, 1995). The steps for the
latter were first fully deciphered by M.Calvin, A. Benson and J.
Bassham at the University of California at Berkeley, for which
Calvin was rewarded with a Nobel Prize in Chemistry in 1961. This
aspect of photosynthesis will not be included in my chapter (see
e.g., Bassham et al., 1954; Calvin, 1989 for the Calvin cycle;
and Cleland et al., 1998, for the current understanding of the
mechanism of the function of its key enzyme RUBISCO). Later,
through the work of Kortsckak et al. (1965), Karpilov (1960) and
Hatch & Slack (1966), it was discovered that in certain
plants (such as sugarcane), the first product of CO2 fixation is
a 4-C organic acid (e.g., oxaloacetic acid) in contrast to what
Calvin and co-workers had found in the green alga Chlorella
where the first stable product ofCO2 fixation was a 3-C
organic acid phosphoglyceric acid. This C-4 pathway is an
appendage to the Calvin cycle (C-3 pathway) in plants such as
sugarcane and pineapple. The discovery of the C-4 pathway has
been elegantly presented by Hatch (1992). An important
consideration about photosynthesis must be mentioned here: almost
complete photosynthesis, i.e., CO2 fixation, occurs within the
chloroplast as first convincingly shown by Arnon (1955).

THE
CHLOROPHYLLS

The most visible colour on our Earth is green, a colour quite
pleasing to our eyes. Nature evolved chlorophylls to capture
sunlight; their structures were such that they could absorb blue
and red light, and transmit green light giving leaves the colour
green; nature also evolved rhodopsin in our eyes for us to see
efficiently this green light. A major landmark in photosynthesis
research was the elucidation of the physical and chemical
properties of the chlorophyll molecule, a tetrapyrrole that has
conjugated (alternating) single and double bonds giving it the
spectroscopic properties of absorbing blue and red light, and a
unique chemical structure allowing it to have different binding
properties to different set of amino acids in various proteins to
make it into light absorbing antenna, as well as reaction center
chlorophylls labelled as P680 or as P700 (P for pigment trap, the
numbers indicating the wavelength maxima of the first singlet
excited state, in nanometers). Only P680+/P680 was destined to
have the redox potential so positive (Em,7 :~ +1.1 V) that it can
oxidize water to molecular oxygen (average Em,7 +0.8 V), whereas
P700+/P700 (Em,7 +0.4 V) is only able to oxidize intermediates
such as the hemes in cytochromes or copper in plastocyanin.

The elucidation of the chemical properties and the structure
of chlorophylls was rewarded with the Nobel prizes in Chemistry
to Richard Wilstätter (in 1915), to Hans Fischer (in 1930),
whereas its total synthesis was recognized with a Nobel prize,
also in Chemistry, to R. Woodward (in 1965). It wasDuysens
(1952) who had first used the term "P" for pigment
representing a few reaction centre chlorophyll a or
bacteriochlorophyll molecules in his Ph D thesis at the State
University of Utrecht. The term P870 for bacterial reaction
centre was then coined. Kok (1956, 1957), who was then at the
Wageningen Agriculture University's 'ship-shaped' building,
discovered P700, the reaction centre chlorophyll of what we now
call Photosystem I (PSI, see a later section), whereas it was
H.T. Witt and coworkers in Berlin (see Döring et al., 1969) who
discovered P680, the reaction centre chlorophyll of what we now
call Photosystem II, PSII. I note that my mentor and second
advisor E. Rabinowitch had long suspected its presence, and
Rabinowitch & Govindjee (1965) had named it as such prior to
its discovery.

Although P680 and P700 are "special pairs" of
chlorophyll a molecules, they are entirely different
mainly because of their binding to different but specific amino
acids in PSII and PSI reaction centre proteins. There are other
chlorophylls too: Chl b, Chl c, and Chl d.
In spite of my first advisor R. Emerson's early speculative and
tentative ideas, Chl b and Chl c are only antenna
pigments, i.e., they function only as light-harvesters in plants
(as well as in green algae & prochlorophytes) and brown algae
(as well as in diatoms & dinoflagellates), respectively. The
recognition of Chl d as the major antenna pigment in Acaryochloris
marina, a prochloron like prokaryote was discovered by
Miyashita et al.

(1996). Whether Chl d can serve as the reaction centre
Chl is under investigation in Japan. In addition, the possible
role of Zinc chlorophylls versus Magnesium chlorophylls is also
being actively investigated by S. Itoh and coworkers (personal
communication).If confirmed, the latter two would break
the stereotypic knowledge we had thus far regarding the
uniqueness of Chl a as the only reaction centre
chlorophyll of oxygenic photosynthesis.

The importance of the chlorophylls has been recognized by at
least two books, edited by Vernon & Seely (1966) and by
Scheer (1991). And finally, the recent discovery of a new
type of bacteriochlorophyll, labelled as bacteriochlorophyll g
(BChl g) (see Gest & Favinger, 1983; Brockman &
Lipinski, 1983; Figure 1; also see Amesz,
1996) may have completed the discovery of all the various types
of chlorophylls present in nature. BChl g resembles BChl b
in having an ethylidene group on C8, which in the presence of
oxygen and light, isomerises to give a vinyl group. Further, BChl
g has a second vinyl group on C3; the product of its
isomerisation is a molecule which is very similar to Chl a,
the pigment of plants and green algae. Thus, heliobacteria
acquire a possibly new evolutionary significance.

The key character of Chl a is that its bound forms,
P680* and P700*, are photoenzymes
converting light into chemical redox energy within a few
picoseconds, acting as the world's most efficient solar battery:

(uphill electron transfer overcoming about 1.1 electron volts
of energy barrier by a far-red photon; Ao is a chlorophyll
monomer bound at a specific site in Photosystem I)

PHOTOSYNTHETIC
UNIT: ANTENNA AND REACTION CENTRES

The terms antenna and the reaction centres have already been
alluded to above. The concept is simple: a large number of light
harvesting molecules function to capture light energy and act as
if they are "antennae"; this captured light energy, in
the form of excitation energy (or excitons) is transferred to a
few pigment molecules that serve as reaction centre pigment
molecules where primary photochemistry takes place. By primary
photochemistry, we mean conversion of light (or excitation)
energy into redox chemical energy that is then stored to do
useful work. The birth of this concept, i.e., of a photosynthetic
unit, that includes the two components, the antenna and the
reaction centre, took place in 1932 in the Kirchhoff Laboratory
of Biological Sciences, at the California Institute of Technology
in Pasadena, CA. There were two players: assistant Professor of
Biophysics Robert Emerson (see Rabinowitch, 1961) and an
undergraduate student William Arnold. Emerson & Arnold
(1932a,b)discovered that under the most optimal condition
of photosynthesis (single turn-over brief and saturating flashes
of light, with optimal dark times between them), a maximum of
only one oxygen molecule was evolved per about 2,400 chlorophyll
molecules present in the green alga Chlorella (Figure 2) although the maximum quantum yield
of oxygen evolution (i.e., the number of O2 evolved per quantum
absorbed) must have been very high, 1/8 to 1/10 in today's
numbers). Thus, the existence of a Unit (and a photoenzyme) was
suggested. It was, however, Gaffron & Wohl (1936) who
provided the correct and complete interpretation of the highly
elegant and sophisticated experiments of Emerson & Arnold:
light absorbed by most of the chlorophyll molecules in the
"photosynthetic unit" is transferred to the
"photoenzyme" for chemistry. The 1932 experiments were
unique in another sense: they were the first experiments in
Science to use repetitive flash technique increasing the
Signal/Noise ratio allowing precise measurements of small
quantitities of oxygen evolution by manometric methods. It is of
historical interest to mention here that both the techniques of
manometry and the green alga Chlorella, used in this work,
were introduced to Science by Emerson's own Professor Otto
Warburg, who had received the Nobel prize in Physiology &
Medicine in 1931 for his studies on biochemistry. The 1932 papers
of Emerson & Arnold are now classical papers and continue to
be regularly cited even after 66 years of their publication. This
milestone discovery was recently discussed in an elegant paper by
Myers (1994), in a special issue of "Photosynthesis
Research", edited by Govindjee et al. (1996),and in
a recent book byWild & Ball (1997). The
Emerson-Arnold "Photosynthetic Unit" has a functional
definition : about 2,400 chlorophyll molecules cooperate to
evolve one oxygen molecule (and, thus, reduce one CO2 molecule).
Asnoted above, photosynthetic unit includes antenna
complexes (the light harvesters) and the reaction centres.
Excitonmigration occurs in femto-to- picosecond range,
among the photosynthetic pigments located in the protein
complexes, the antenna proteins (see Hoff & Amesz, 1995; Hoff
& Deisenhofer, 1997). The first kinetic evidence for the
excitation energy transfer was obtained from time-resolved
ultrafast fluorescence spectroscopy experiments initiated by
Brody & Rabinowitch (1957)at UIUC, Urbana, Illinois.
A wonderful example of this was shown by Yamazaki et al.(1984)in red algae where one can literally watch the excitons move
from phycoerythrin to phycocyanin, then to allophycocyanin, and
finally to chlorophyll a in a cascade-like manner.

Major breakthroughs in the understanding of the antenna
structure at the atomic level have been through the availibility
of X-ray and electron diffraction crystallography. Kühlbrandt et
al.(1994) have provided the atomic level structure of the largest
antenna of Photosystem II, the Light-harvesting complex II b;
here one can see where the Chl a and Chl b molecules
are anchored. On the other hand, McDermott et al. (1995) and Walz
and Ghosh (1997) have provided the atomic level structures of two
light-harvesting antenna complexes of anoxygenic photosynthetic
bacteria; and, Hofmann et al. (1996) have provided the atomic
level structure of the peridinin-chlorophyll protein complex of
the dinoflagellate Amphidinium carterae. These provide the
most fascinating view of the grand design of the architecture of
the photosynthetic apparatus responsible for the capture and
trapping of the excitation energy. These newer structures had
been preceded by the structure of the chlorobium protein complex
(the so-called Fenna-Mathews-Olson protein, see Fenna &
Mathews, 1976; Mathews & Fenna, 1980; Tronrud et al., 1986),
of phycoerythrin (Fiener & Huber, 1993) and of phycocyanin
(Schirmer et al., 1986). Thus, we are now able to ask meaningful
questions about the detailed physico-chemical reactions in the
antenna and finally begin to understand the molecular mechanism
of excitation energy transfer in photosynthesis, an area that had
been dominated in the past mainly by speculative and theoretical
arguments.

The conversion of light energy into chemical energy occurs in
the World's most efficient solar battery, in the picosecond time
range, in the reaction centre molecules. This conversion leads to
the formation of the primary charge-separated state, P+A-, from
PA, where P is the special reaction centre molecule, as already
mentioned, and A is the primary electron acceptor. Examples of
this reaction were already given under the section CHLOROPHYLLS.
This is the only true light reaction of photosynthesis; all
others can, in principle, occur in darkness. They are the only
ones that are "uphill" because the free energy is
positive due to the unfavourable difference in redox potentials
of P/P+ and A/A-. These primary reactants and those involved in
charge stabilization are located in the reaction centre
complexes. The X-ray diffraction crystal structure of the
reaction centre complex of the anoxygenic photosynthetic
bacterium Rhodopsuedomonas viridis was the first one to be
published, and was rewarded with the 1988 Nobel prize in
Chemistry to H. Michel, J. Deisenhofer and R. Huber (see
Deisenhofer et al., 1984,1985, 1995; Feher et al., 1989; Feher,
1998).Recently,Rhee et al. (1997) have provided a
crude structure of P680-containing Photosystem II reaction centre
complex at 8 Å; there are no details available. However,
knowledge-based atomic level models of Photosystem II are now
available (see e.g., Xiong et al., 1996, 1998; see Figure 3 and its legend ; also see a partial
model by Coleman et al., 1997). Just as
femtosecond-to-picosecond absorption spectroscopy have provided
information on the primary charge separation in anoxygenic
photosynthetic reaction centre (see e.g., Hoff & Deisenhofer,
1997; Zinth et al., 1998),information on Photosystem II
are also now available (see Greenfield and Wasielewski, 1996; and
Groot et al., 1997). Greenfield et al. (1997) have suggested that
at ambient temperatures two time constants of charge separation
can be measured:(1) approximately 8 ps, an upper limit for charge
separation, due in part to equilibration of excitons among the
core reaction centre chlorophylls (the "red" pool, R);
and (2) approximately 50 ps, due, in part to equilibration of
excitons among the accessory chlorophylls (the "blue"
pool, B).

We believe that the actual charge separation time is closer to
3 ps, the same as in anoxygenic photosynthetic bacteria. (In the
equations shown above, * represents the molecule with an exciton
or a photon.) All of the experiments on the primary
photochemistry of photosystem II have been possible because of
the success of Nanba & Satoh (1987) in isolating the
simplest, although incomplete, reaction centre protein of
photosystem II.

In contrast to PSII, a 6 Å resolution structure of
P700-containing Photosystem I (PSI) reaction centre was published
already five years ago by the research group of H.T. Witt of
Berlin (Krauss et al., 1993); now, a 4 Å resolution structure of
the same complex is available (Krauss et al., 1996). In addition
to differences in the details, e.g., PSI being a Fe-S containing
reaction centre, the PSI reaction centre complex contains also
the core antenna pigment as well as the antenna chlorophylls. I
consider this work to be one of the major milestones in the
history of photosynthesis.

THE TWO PIGMENT
SYSTEMS AND THE TWO LIGHT REACTIONS

Electron transfers occur in pico-to-millisecond time range;
they involve, in oxygenic photosynthesis, three major protein
complexes: Photosystem II (water-plastoquinone oxido-reductase);
Cytochrome b6/f complex (plastoquinol-plastocyanin
oxido-reductase); and Photosystem I (plastocyanin-ferredoxin
oxido-reductase). A fourth ATP synthase is required for ATP
synthesis (see Figure 4 and its legend).
An enigma, discovered by Emerson & Lewis (1943), was that the
quantum yield of oxygen evolution decline suddenly beyond 680 nm,
when chlorophyll a was still absorbing light; this
so-called "red-drop" phenomenon led to the current
concept of the two light reactions and two pigment systems when
Emerson et al. (1957) discovered the Enhancement effect in
photosynthesis. This concept is based on solid grounds: from the
early ideas of E. Rabinowitch (1945, p.162; 1956, p. 1862) and of
Hill & Bendall (1960). In the experiments on the so-called
Emerson Enhancement in photosynthesis (see Figures
5 & 6 and their legends), or in
the Hill Reaction, yield in far-red light (light I) by
simultaneous exposure to shorter wavelength light (light II), is
enhanced (Emerson et al., 1957; alsosee Emerson, 1958;
Emerson & Rabinowitch, 1960; Govindjee & Rabinowitch,
1960; R. Govindjee et al., 1961, 1962, 1964; and a review by
Myers, 1971). Further, the crucial experiments on the
antagonistic effect of light I and II on the redox state of P700
(Kok, 1959; see Figure 7 and its legend),
on the Chlorophyll a fluorescence yield (Govindjee et al., 1960;Butler, 1962; Duysens & Sweers, 1963), and, most
importantly on the redox state of cytochrome f (Duysens et al.,
1961; see Duysens, 1989) established the two light reaction
scheme of photosynthesis. In fact, the existence of the series
scheme of photosynthesis is best proven by light I (absorbed in
photosystem I) oxidising cytochrome f and light II (absorbed in
photosystem II) reducing the oxidised cytochrome f (see Figure 8 and its legend). However, without
the fast absorption spectroscopic work on the new intermediate
X-320 (now known to be equivalent to the so-called QA, the first
bound plastoquinone molecule), the history of the discovery of
the two light reactions would be incomplete (see Witt et al.,
1961; Witt, 1991). Figure 9 shows the
current model of the so-called Z scheme of electron transfer in
oxygenic photosynthesis, whereas the legend of Fig.9 describes
the current scheme. (The reader should refer to Figure 4 for the
location of the components described in this figure.)

Electron
transport: General

The mechanism by which electrons are transferred from one
molecule to another has now been explained by R. Marcus (1993)
who was awarded the 1992 Nobel prize in Chemistry for it..
A brief discussion follows. When a system such as e.g., DA is
converted into D+A-, i.e., an electron is transferred from D to
A, the energy curves of the two states DA and D+A- are shifted
with respect to each other on axis of the reaction path (see Figure 10). Marcus defined a new term
called the reorganizational energy (l ) by the length of the
vertical line from the energy curve of the DA state to where the
line hits the D+A- curve. The rate constant of the reaction is
given by:

k= A exp ( -D G*/kBT) (6),

where A depends on the nature of the electron transfer
reaction, kB is the Boltzmann constant, T is the absolute
temperature in Kelvin, and D G* is the activation free energy for
the reaction. And,

D G* = l /4 (1+ D Go/l )2 (7),

where D Go is the standard free energy for the reaction, and l
is the reorganizational energy, as mentioned above. A major
prediction of the Marcus equation (7) is that as D Go is varied from zero to some
negative value, D G* decreases and
becomes zero at D Go = -l , then increases when D
Go is made still more negative , i.e., -D Go > l
. The initial decrease of D G* with
increasingly negative D Go
was as expected, but what Marcus equation explained was the rate
of electron flow in what he had called the "inverted
region": increases in D G* when D Go was made increasingly
negative. This prediction was made in 1960, but was confirmed
experimentally only in 1984 by G.L. Closs and coworkers. Marcus
(1993) has extended his theory to explain the characterestics of
electron transfer in the bacterial reaction center. It is
interesting to point out that electron transfers during the
primary charge separation, discussed earlier, do not slow down at
cryogenic temperatures; in fact, they become temperature
independent. Such a phenomenon had been discovered by DeVault
& Chance (1966) (also see DeVault, 1989): it was suggested
that electrons can "tunnel" through barriers as if they
squeeze through with certain probability. Further, if the
energetics and other entropic parameters are constant, electron
transfer rates can be predicted from the distances of the
acceptor from the donor molecules. Moser et al. (1992) showed
that if the acceptor is 1.4 Å further away, the transfer rate
decreases by an order of magnitude. This, known as the
"Dutton ruler", does not explain why electrons flow
only on one side of the bacterial reaction centre molecule, not
the other. Future prospects of understanding the mechanism of
electron transfer are increasing as we begin to have atomic
structures of the various intermediates. For example, we now have
access to atomic level structures of the hydrophilic parts of
cytochrome f (Martinez et al., 1994); of plastocyanin (Redinbo et
al., 1993); and of the Cyt b/ccomplex of beef heart
mitochondria (Xia et al., 1997) that may have similarities to Cyt
b6/f complex of plants. Zhang et al. (1998) have described a
model of electron transfer in which movement of domains occurs in
cytochrome b/c1 complex; I expect it to have application to
electron transfer in cytb6/f complex. However, in spite of the
available structure of the reaction centre of anoxygenic
photosynthetic bacteria, which have tremendous similarities in
their two halves, we still do not fully understand why the
electrons flow mostly on one, rather than the other side of the
molecule. Here, a combination of molecular biology and physics
& physical chemistry have begun to play important roles in
the understanding of this process. We are still waiting for the
final understanding.

Electron
Transport: Acceptor Side of Photosystem II

A major advancement took place in the understanding of the
functioning of the electron transfer pathway when Velthuys &
Amesz (1974) and Bouges-Bocquet (1973) showed that there is a two
electron gate on the acceptor side of PSII (see Figure 11 and its legend); a
similar gate was discovered later in anoxygenic photosynthetic
bacteria, independently by Vermeglio (1977) and by Wraight
(1977).Electrons from the reaction centers are
transferred first to a bound quinone molecule QA which is,
surprisingly, a one-electron acceptor. The reduced QA transfers
its electron to a loosely bound quinone QB; the reduced QB is
tightly bound and has a long lifetime; it waits for a second
electron until it can be doubly reduced to QB2-. The latter then
becomes a quinol (QBH2) after grabbing two protons coming from
the side of the membrane it is located on. The electron flow at
the QAQB site can be written as (see a review by Crofts &
Wraight, 1983):

QAQB+ light-------> QA -QB-----> QAQB- (8)

QAQB- + light ------> QA QB2- (9)

QA QB2-+ 2H+------> QA QBH2 (10)

QA QBH2 + PQ-------> QAQB + PQH2. (11)

Although it is not certain, especially in plants, whether the
protonation steps occur before the electron flow or vice versa,
the above steps are referred to as the "two electron
gate".

The bicarbonate
effect.I have had a personal interest in this
two electron "gate". Over the past twenty-five years or
more, we have been trying to understand the role of bicarbonate
in the reactions on the two photosystems, a phenomenon discovered
by Warburg & Krippahl (1960). Warburg (1964) took this
phenomenon to suggest that oxygen in photosynthesis originated in
CO2, not in water, contradicting the existing idea that oxygen
originated in water. He argued that (1) the 18O experiments of
Ruben et al. (1941) could not be trusted because of equilibration
with both water and CO2; (2) the hypothesis of C.B. van Niel was
only from comparative biochemistry between oxygenic and
anoxygenic organisms, and thus had no definitive value; and (3)
the requirement of bicarbonate in the Hill reaction showed that
it cannot be taken to prove that CO2 was not the source of oxygen
in photosynthesis. I note here parenthetically that the discovery
of the Hill reaction (see Hill & Scarisbrick, 1940), i.e.,
reduction of an artificially added electron acceptor and
evolution of oxygen by chloroplasts, was itself a major finding
that allowed tremendous number of biochemical studies that ensued
from that time; it opened the field to look under the
"hood" of the car, so to say. These were the arguments
that fascinated me. I presented them to a photosynthesis course I
was teaching in the early seventies; it was attended, among
others, by Prasanna Mohanty and Alan Stemler, my PhD students.
Alan jumped at the idea of working on the exciting project of
attempting to understand the role of bicarbonate in the Hill
reaction. Our first paper (Stemler & Govindjee, 1973)
suggested that bicarbonate may function on the water side of
photosynthesis. However, very soon we discovered a major effect
on the acceptor side of photosystem II. We located a role on the
electron acceptor side, specifically at the two-electron gate
(Wydrzynski & Govindjee, 1975; Govindjee et al., 1976;
Jursinic et al., 1976; Khanna et al., 1977, 1981; Blubaugh &
Govindjee, 1988; Eaton-Rye & Govindjee, 1988; Xu et al.,
1990; Govindjee, 1993, and, Govindjee & van Rensen, 1993).
Bicarbonate seems to be involved not so much in the electron flow
per se, but in the protonation steps (also see van Rensen
et al., 1988). Interestingly, this effect is absent in anoxygenic
photosynthetic bacteria (see e.g., Shopes et al.,.1988; Wang et
al., 1992). Differences between photosynthetic bacteria and
photosystem II are, we believe, due to a preponderance of
positively charged amino acids in the QB binding region of PSII
over that in photosynthetic bacteria (Xiong et al.,1996;
1998a). This may be responsible for different protonation
channels in anoxygenic photosynthetic bacteria and in PSII. This
bicarbonate site seems close to (Khanna et al., 1981) , but not
identical to that where certain herbicides bind and act
(Velthuys,1981;Wraight ,1981). Terbutryn inhibits and
kills plants by displacing QB, and bicarbonate seems to be
involved in the protonation of the reduced form of QB. One of the
binding sites of bicarbonate has been shown to be on the non-heme
iron ( see e.g., Diner et al., 1991) and the other near an
arginine residue, D1-R-257 (Xiong et al., 1998b). It is not yet
clear how this acceptor side binding (see Figure
12) would provide another major bicarbonate action on the
oxygen evolving complex expounded by O. Warburg, H. Metzner and
A. Stemler(see Stemler, 1982),and now being
investigated in great detail by Klimov & coworkers
(Allakhverdiev et al., 1997; Hulsebosch et al., 1998; Klimov et
al., 1997; Yruela et al., 1998). It calls for further research.

Oxygen
Evolution

The answer to the question of the minimum number of quanta
needed to evolve one oxygen molecule was solved in favour of 8-10
(Emerson & Lewis, 1943; Emerson, 1958; R. Govindjee et al.,
1968) rather than of 3 to 4 (Warburg & Negelein, 1923). This
is in agreement with the scheme that two light reactions are
needed for oxygen evolution and NADP+ reduction. A major
breakthrough in the oxygen evolution steps was the discovery by
Joliot et al. (1969) that oxygen/flash as a function of flash
number oscillates with a period of 4 indicating accumulation of 4
positive charges on some intermediate before water is oxidized to
molecular oxygen (Figure 13). A theory
evolved by Kok et al.(1970) became the major framework (for
alternate models, see Mar & Govindjee, 1972), where the
system started mostly in the S1 state in darkness, and oxygen
release took place when S4 was converted to S0 (S representing
the redox state of the tetranuclear Mn complex):

` S1 + light flash #1; or #5---> S2 (12)

S2 + light flash # 2; or #6---->S3 (13)

S3+ light flash # 3; or # 7----> S4 (14)

S4 + 2H2O-----> S0+ O2 + 4 H+ (15)

S0 + light flash # 4; or # 8---> S1. (16)

The elegant measurements of Dismukes & Siderer (1980) on
multiline EPR (electron paramagtnetic resonance) signals of Mn,
and those of M. Klein and coworkers (see Yachandra et al., 1996;
Roelofs et al., 1996) on EXAFS (Extended X-ray Absorption Fine
Structure) and XAS ( X-ray Absorption Spectroscopy) led to the
identification of the S-state intermediates with mixed valence Mn
complex (a tetranuclear Mn/PSII). The S0 is suggested to be Mn
(II), Mn (III), Mn (IV), Mn (IV) (alternatively, Mn (III), Mn
(III), Mn (III), Mn (IV)); S1 to be Mn (III), Mn (III), Mn (IV),
Mn (IV); and both S2 & S3 as Mn (III), Mn (IV), Mn (IV), Mn
(IV), Mn (IV). The mechanism of water oxidation to
dioxygen is not yet known; however, there are theories as to how
this occurs. I refer the readers to read Renger (1997) and
Babcock & co-workers (Hoganson& Babcock,1997; Tommos
& Babcock, 1998) to obtain a glimpse of the current and two
different thoughts on the mechanism of oxygen evolution.The
recent finding of Messinger et al. (1997a) that S0 is also
paramagnetic will further enhance our ability to understand the
transitions of the S states, presented above. In addition,
the possibility of showing that the S states can be reduced to
S(-3) state lends credence to the current picture of the valence
state of the Mn in the water oxidase (see Messinger et al.,
1997b). On the personal side, I want to mention that Coleman
& Govindjee (1987) were the first to suggest that the Mn
atoms of the Mn cluster were bound to the reaction centre
proteins D1 & D2; and, Kambara et al. (1985) and Padhye et
al. (1986) were the first to suggest that an organic radical,
specifically a histidine, may be the charge accumulator in
addition to Mn during S2 to S3 transition. The role of histidine
is being actively investigated in several laboratories; first, it
seemed evidence was obtained, then, it was challenged; now, I am
waiting for the final judgement before citing any paper on this
topic. Following the early pioneering work of W.F.J. Vermaas and
of B. Diner and coworkers, Bowlby et al. (1996) have reported
that Glutamic-65 and Histidine 337 on D1 protein may be ligands
to Mn; and, Aspartic 103 and Glutamic 104, also on D1, may be
ligands to Calcium. On the other hand, T. Wydrzynski and
coworkers (Messinger et al., 1995) are providing new information,
using elegant mass spectroscopic methods, on the exchangeable
water molecules at the site of water oxidation. Evidence for the
proximity of a Calcium to the Mn cluster, discussed above, has
now been implied from X-ray absorption spectroscopy (Latimer et
al., 1995). Oxygen, the by-product of water oxidation, is
released through the operation of the "oxygen clock"
that involves 4 Mn atoms/PSII (see a detailed earlier review by
Debus, 1992).

Thermoluminescence

A major tool to study the S-states was shown to be
thermoluminescence, a phenomenon discovered by W. Arnold ( see
reviews by Sane and Rutherford, 1986; Vass & Govindjee, 1996).
Thermoluminescence (TL) is light emission, that originates mainly
from PSII. If one shines light on a photosynthetic material, and
freezes to 77K, all the separated charges are frozen, and heating
the sample in darkness leads to thermoluminescence bands
originating from the back reactions (i.e., recombination of
charges) of say, e.g., S2 with QB- or S2 with QA-, etc. P.V.
Sane, in collaboration with V.G. Tatake and T.S. Desai, was
responsible for placing India on the World map of pioneering
research in this area of thermoluminescence. The design of the
instrument at the Bhabha Atomic Research Centre (BARC), Mumbai,
India, where this work was done, provided very high Signal/Noise
ratio. Several (6 or 7) TL bands were fully separated from each
other and these bands, through separation of pigment systems I
and II and through the ingenious use of inhibitors and electron
carriers, were related to specific reactions in PSII and even PSI
(see Sane & Rutherford, 1986; and Vass & Govindjee, 1996,
for reviews). It was because calculations, by Tatake et al.
(1981), of the activation energies of the various TL bands did
not match the Randall-Wilkins theory that I approached Don
DeVault for help; this led to the formulation by DeVault et al.
(1983) of the most appropriate theory for TL in plants. Without
the leadership provided by P.V. Sane, researches on TL at BARC
would have been in directions different from those on the
photosynthetic systems. Relationship of the S-states to the TL
was, however, provided byInoue &Shibata (1978) when
they discovered the period 4 oscillation in TL and related it to
oxygen evolution steps (see reviews by Vass & Inoue, 1992;
and Inoue, 1996). I refer the readers to the cited reviews to
discover for themselves the discoveries of A.W. Rutherford, G.
Renger, and the research group in Szeged, Hungary without whose
work, TL will not be where it is today. Currently, the TL method
is being used to monitor and characterise site-directed mutants
both on the acceptor (see e.g., Mäenepää et al., 1995) as well
as the donor side of PSII (see e.g., Kramer et al., 1994).

PHOTOPHOSPHORYLATION

ATP is the energy currency of life. Production of glucose
(food) from CO2 requires both NADPH and ATP. Below, we will
discuss the breakthroughs that led to the understanding of ATP
synthesis: chemiosmotic hypothesis; and the alternate binding
site hypothesis.

After indications in several laboratories that algal cells
produce ATP in light, Arnon et al. (1954) and Frenkel (1954)
discovered photophosphorylation in chloroplasts of plants, and
chromatophores of anoxygenic photosynthetic bacteria,
respectively. This was a major breakthrough and it took many
years to recognize that only in some anoxygenic
photosynthetic bacteria, almost all light energy is first
converted into ATP energy and then this energy is used for the
reversed electron flow to produce the reducing power in the form
of NADH. In oxygenic organisms, however, this is not the case as
correctly expressed by Rabinowitch (1956). As we know today, ATP
synthesis follows electron transport steps that first store
energy temporarily by creating a proton motive force; this energy
would be otherwise lost if not used for ATP formation (see the
scheme of Hill & Bendall, 1960). P. Mitchell (1961), who
received the Nobel prize in Chemistry in 1978, provided the
theory that a proton motive force ( that is a sum of a pH
gradient and a membrane potential) is the energy source of ATP
synthesis. Membranes are normally impermeable to protons; and,
protons are transferred from one side of the membrane to the
other by virtue of the alternate electron and hydrogen atom
transfers due to specific location of the electron and hydrogen
atom carriers and the energetics of the light-induced electron
transfers in photosynthesis. An early breakthrough was the
independent experiment of Shen & Shen (1962) and of Hind
& Jagendorf (1963) showing that light forms some entity (XE)
that can be used later in darkness to produce ATP. Hind &
Jagendorf showed that XE is a pH gradient that drives
ATP synthesis. Jagendorf & Uribe (1966) showed that ATP can
be synthesized from pH gradient created by acid-base transition
in total darkness. These experiments, along with those of the
others, provided major evidence for the chemiosmotic hypothesis
of Mitchell. In photosystem II, water oxidation complex that
liberates protons is on the inner side (the lumen) of the
thylakoid membranes. Further, plastoquinone is reduced to
plastoquinol on the outer side (stroma side) of the thylakoid
membrane; the plastoquinol (a hydrogen atom carrier) moves
towards Rieske Iron sulfur and cytochrome f (both are electron,
not hydrogen atom carriers) that are located towards the lumen
side. Here, plastoquinol delivers its electrons to the Rieske
Iron sulfur centre and the cytochrome f, leaving the protons to
be released into the lumen. This arrangement, thus, allows
natural proton translocation from the stroma to the lumen side as
the electron transport takes place in PSII. This adds to the pH
gradient to be used for ATP synthesis. The equivalence of pH
gradient and membrane potential in synthesizing ATP was shown by
the elegant experiments of Gräber et al. (1984) and Hangarter
& Good (1982)when they varied one keeping the other
constant and showing that it was the sum of the two that was
important for initiating ATP synthesis.

The understanding of the mechanism by which ATP synthesis
takes place at the ATP synthase using the proton motive force has
been influenced by the following three breakthroughs. (1) The
binding change hypothesis of P. Boyer and co-workers (see Boyer
et al., 1973; and a review by Boyer, 1997; also see Figure 14 and its legend) that suggests that at
one time (a) ADP and Pi (inorganic phosphate) bind weakly at one
site of the alpha-beta pair of the F1 part of the ATP synthase
enzyme; (b) bound ATP is formed from bound ADP and Pi, without
the use of energy at a second alpha-beta pair; and (c) the pH
gradient energy is converted into rotational energy, mainly of
the gamma subunit that extends upto the third alpha-beta pair,
that is used to flip off the ATP free from the third alpha-beta
binding site. These three sites alternate in time. (2) The
rotation feature of the Boyer model has been elegantly
demonstrated directly by fluorescence microscopy and by
photoselection and other experiments (see Capaldi, R.A. 1994;
Duncan et al., 1995. Sabbert et al., 1996, 1997; Junge et al.,
1997) (3) Atomic resolution structure (Abrahams et al., 1994) of
beef-heart mitochondrial F1 showed that the gamma subunit indeed
looks through the alpha-beta pairs; and in agreement with the
Boyer model, the structure shows one alpha-beta pair site empty
(as if ATP was released); another with bound ADP and Pi; and the
third with an equivalent of bound ATP. I consider it highly
likely that the plant, algal, and cyanobacterial ATP synthase
will be basically similar to that of the cow.

CONCLUDING REMARKS

In my opinion, the mechanism of photosynthesis has been probed
by several means. Its face has changed by experiments of many
investigators. I restrict my personal list to deal mainly with
oxygen evolution. It includes, among others, the experiments of
my first mentor Robert Emerson (Emerson & Arnold,1932). U
sing the repetitive flash method and Warburgs manometry,
they led to the concept of "Photosynthetic Unit" where
excitation energy, absorbed by hundreds of antenna pigment
molecules, is transferred to the reaction center chlorophyll
molecules, the "photoenzyme" or the "energy
trap" for chemistry. Next, was the discovery of the
"Red Drop" (Emerson & Lewis, 1943) in the quantum
yield action spectrum of oxygen evolution in the green alga Chlorella
pyrenoidosa, and the enhancement effect of certain
wavelengths of light on the yield of oxygen evolution in the
"red drop" region (Emerson et al., 1957), discovered by
the use of an excellent monochromator and state-of-the art
manometry; it lead to the concept of two-light reaction and
two-pigment system mechanism of electron transport. It became
well-known due to the working hypothesis of Hill & Bendall
(1960) and became a scientific fact by the experiments of Duysens
et al. (1961) on the antagonistic effect of light absorbed in
pigment system I and II on the redox state of cytochrome f.
The important concept that there is an "oxygen clock",
where four positive charges must accumulate before water can be
oxidized to oxygen was enshrined before us by (a) the experiments
of Joliot et al. (1969) on the periodicity of four in the plots
of the amount of oxygen evolved per flash as a function of flash
number, and (b) the so-called "S-states" model of
charge accumulation enunciated by Kok et al. (1970). Such a
periodicity of four, reflecting indirectly the S-states, has also
been observed in chlorophyll a fluorescence (Delosme,
1971; Joliot & Joliot, 1971), and recently, Shinkarev et al.
(1997) have even managed to obtain the kinetics of the last step
of oxygen evolution from analysis of the data on the decay of
chlorophyll a fluorescence in single flashes of light.
Thermoluminescence, discovered by W. Arnold & H. Sherwood
(see Arnold, 1991), that has been exploited both by P.V. Sane
(Sane & Rutherford, 1986) as well as myself (DeVault et al.,
1983; Vass & Govindjee, 1996), has uniquely probed the
characteresteristics of the S-states of oxygen evolution (Inoue
& Shibata, 1978; Inoue, 1996). The most elegant probes for
showing that Koks S-states are Manganese are the low
temperature EPR (Dismukes& Siderer, 1980); and the Extended
X-ray Absorption Fine Structure (EXAFS) spectroscopy (M. Klein,
K. Sauer & co-workers; see Yachandra et al., 1996). At the
end, I do want to emphasise two elegant conceptual work on
another area, that of phosphorylation: (a) the chemiosmotic
theory of P. Mitchell in which proton motive force across a
membrane provide energy for ATP synthesis (Mitchell, 1961) and
(b) the elegant theory of Paul Boyer as to how this comes about:
by conversion of electrochemical energy to conformational energy
(see Boyer, 1977). Finally, I wish to mention that mimicking
photosynthesis in vitro has been a dream of many, and the
recent success of Steinberg-Yfrach et al. (1998) in making
"lots of ATP" in artificial liposome membranes,
energised by a synthetic system (carotene-porphyrin-quinone, is
highly commendable. It would have made my second mentor Eugene
Rabinowitch quite happy.

Duysens, L.N.M. & Sweers, H.E. 1963. Mechanism of the two
photochemical reactions in algae as studied by means of
fluorescence. In Japanese Society of Plant Physiologists (eds) Studies
on Microalgae and Photosynthetic Bacteria:: 353-372.
University of Tokyo Press, Tokyo.

Kramer, D., Roffey, R.A., Govindjee & Sayre, R.T. 1994.
The At thermoluminescence band from Chlamydomonas reinhardtii and
the effects of mutagenesis of histidine residues on the donor
side of the photosystem II D1 polypeptide.